We propose that extracellular vesicles (EVs) can not only act as delivery vehicles for growth factors in therapeutic applications, but they also represent a novel growth factor signaling pathway to affect therapeutic outcomes. EVs are present throughout the body and are involved in intercellular communication, locally and systemically, from early development onward, and across physiological and pathophysiological conditions. EVs occur as soluble EVs in the extracellular microenvironment surrounding the cell and as solid-phase EVs immobilized within the extracellular matrix (ECM), thus creating EV/ECM microenvironments. EV cargo, such as growth factors, occur either on the EV surface or internalized within the EV lumen. Although growth factors on the surface of EVs appear to signal target cells via cell surface receptors, the signaling mechanisms of EV luminal confined growth factors remains unknown. We challenge long-held endocrine dogma arguing that growth factors, as proteins, must first bind to their surface receptors on target cells as a prerequisite to the initiation of intracellular signaling cascades. We hypothesize that EVs enveloping growth factor luminal cargo form EV/ECM microenvironments, bind to target cells and deliver growth factors directly into the cytoplasm via EV endocytic trafficking, bypassing growth factor cell surface receptors, representing the initiation of an alternative growth factor cell signaling pathway. Our preliminary studies involved loading BMP2 (as a prototypical growth factor) into EVs (BMP2-EVs) and then bioprinted them on ECM scaffolds to engineer BMP2-EV/ECM microenvironments. These BMP2-EV/ECM constructs delivered BMP2-EVs to cells, the BMP2-EVs were internalized, and they induced stem cell differentiation toward osteogenesis in vitro and heterotopic ossification in vivo. The mechanism of BMP2-EV signaling remains to be elucidated because binding to cell surface BMP2 receptors appeared to be bypassed. EV delivery of growth factors offer unique advantages beyond delivery based on ECM-immobilized BMP2 alone, including: protection from growth factor extracellular antagonists, such as noggin in the case of BMP2; synergistic effects of co-delivered native endogenous luminal co-cargo, such as miRNAs; and EV stability during processing and storage. In this R21 we will initiate studies to determine functioning of EV/ECM-delivered growth factors through the following complementary but independent specific aims: Aim 1, Initiate mechanistic studies toward understanding BMP2-EV/ECM signaling in vitro. Aim 2: Conduct animal experiments to determine the ability of implanted biopatterned BMP2-EV/ECM microenvironments to induce bone repair in our standard mouse calvarial critical- size defect (CSD) repair model.